Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Identification of salinity-related genes in ENO2 mutant (eno2~–) of Arabidopsis thaliana

Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR （RT-qPCR） was performed to investigate the expression profile of EN02 in response to NaCl stress in Arabidopsis. The transcript level of EN02 was rapidly elevated in 300 mmol L-1 NaCl treatment. ENO2 also responded to 300 mmol L 1 NaCl treatment at the protein level. To illuminate the mechanism underlying EN02 resistance to salt at the transcriptional level, we studied the wild-type and enoZ Arabidopsis lines that were treated with 300 mmol L 1 NaCl for 18 h using 454 GS FLX, which resulted in an expressed sequence tag （EST） dataset. A total of 961 up-regulated and 746 down-regulated differentially expressed genes （DEGs） were identified in the pairwise comparison w-r-18 h：eno2^-18 h. The DEGs were identified and functionally annotated using the databases of Gene Ontology （GO） and the Kyoto encyclopedia of genes and genomes （KEGG）. The identified unigenes were subjected to GO analysis to determine biological, molecular, and cellular functions. The biological process was enriched in a total of 20 GO terms, the cellular component was enriched in 13 GO terms, and the molecular function was enriched in 11 GO terms. Using KEGG mapping, DEGs with pathway annotations contributed to 115 pathways. The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites （KO01110）, plant-pathogen interactions （KO04626）, and plant hormone signal transduction （KO04075）. Based on these results, EN02 contributes to increased resistance to abiotic stress. In particular, EN02 is involved in some of the metabolic stress response pathways in Arabidopsis. Our work also demonstrates that this EST dataset will be a powerful resource for further studies of EN02, such as functional analyses, investigations of biological roles, and molecular breeding. Additionally, 3-phosphoglycerate kinase （PGK）, 3-phosphoglycerate kinase 1 （PGK1）, triosephosphate isomerase （TPI）, and pyruvate kinase （PK） in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment, and EN02 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that EN02 gene has an important role in the response to the high salt stress.

Comparative analysis of transcriptomes from albino and control sea cucumbers, Apostichopus japonicus

The sea cucumberApostichopus japonicus is an important economic species in China. Its dorsal body wall color is commonly tawny, whereas its ventral surface is fawn. Albino sea cucumbers are rarely observed. In order to profile gene expression and screen albinism-related genes, we compared the transcriptome of albino samples with a control by 454 cDNA sequencing. We found that 6 539 identified genes on the basis of sequence similarity to known genes were expressed in the albino A. japonicus. The gene ontology analysis indicated that the transcription of genes associated with the terms of biological regulation and pigmenta-tion was non-abundant in the albino library compared to the control. Based on an analysis using the Kyoto Encyclopedia of Genes and Genomics （KEGG） database, we identified 14 important genes that were in-volved in major intercellular signaling pathways related to melanin synthesis, such as tyrosine metabolism, the mitogen-activated protein kinase （MAPK） pathway, and melanogenesis. The expressions of fibroblast growth factor receptor 4 （FGFR4）, protein kinase C （PKC）, protein kinase A （PKA）, and Ras genes were sig-nificantly down-regulated in the albino transcriptome compared with the control, while the expressions of homogentisate 1, 2-dioxygenase gene （HGO）, cAMP-responsive element binding protein （CREB）, transcrip-tion factor AP-1（c-jun）, and calmodulin （CaM） were significantly up-regulated （Fisher＇s exact test,p 〈 0.05）. These differentially expressed genes could be candidate genes for revealing the mechanism of albinism and investigating regulation of melanin synthesis inA. japonicus.

Tissue-specific Temporal Exome Capture Revealed Muscle-specific Genes and SNPs in Indian Buffalo(Bubalus bubalis)

Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome （coding region of genome） through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes （TST exome） in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences （up to 40%） and an enrichment of coding sequences （up to 60%）. Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads contain- ing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsyn- onymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.